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[目的]探讨葡萄糖-6-磷酸脱氢酶(G6PD)缺陷对苯醌(BQ)染毒人慢性髓性白血病(K562)细胞毒性的影响。[方法]用不同浓度的BQ(0、10、20、40μmol/L)处理野生型K562细胞,应用MTT比色法检测BQ对受试细胞的增殖抑制作用,Western blot法检测G6PD蛋白表达的变化。构建G6PD-set RNA干扰慢病毒,并感染野生型K562细胞株;以转染空载体的野生型K562细胞作为阴性对照细胞,荧光实时定量-聚合酶链反应(real-time PCR)法检测G6PD基因的m RNA表达量。再用不同浓度的BQ处理G6PD缺陷的K562-WT细胞(K562-G6PD△)和阴性对照细胞,检测细胞增殖抑制作用,比色法检测细胞中还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平。[结果]MTT结果表明,与BQ浓度为0μmol/L组相比,10、20、40μmol/L的BQ作用24、48、72 h后,野生型K562细胞相对增殖率均明显降低(P<0.05)。Western blot结果显示,低剂量下(BQ浓度<40μmol/L时)随着BQ浓度的增加,G6PD蛋白含量亦增加(r=0.809,P=0.008);当BQ浓度升至40μmol/L时,G6PD蛋白含量有所下降,但仍高于BQ浓度为0μmol/L组(P<0.05)。real-time PCR法检测结果显示,K562-G6PD△细胞的G6PD m RNA表达量较阴性对照降低了86.65%,表明K562-G6PD△细胞构建成功。干预G6PD后,MTT结果表明,与阴性对照细胞相比,K562-G6PD△细胞暴露于不同浓度BQ后,其相对增殖率均明显降低(P<0.05)。比色法结果表明,当BQ浓度为20、40μmol/L时,K562-G6PD△细胞中GSH浓度明显降低,而在阴性对照细胞中,当BQ浓度为40μmol/L时GSH浓度明显降低;当BQ浓度为10μmol/L时,较阴性对照细胞相比,K562-G6PD△细胞中GSSG浓度显著升高(P<0.05)。[结论]野生型K562细胞暴露于BQ后细胞增殖受抑制,氧化产物增加的同时可能通过激活G6PD等抗氧化系统以抵抗机体所受的氧化损伤;而G6PD缺陷后,由于G6PD不能被激活,在暴露于较低剂量的BQ时即可使细胞内GSH耗竭导致GSSG在细胞内堆积而使细胞增殖抑制毒性增加。
[Objective] To investigate the effect of glucose-6-phosphate dehydrogenase (G6PD) deficiency on the cytotoxicity of benzoquinone (BQ) -treated human chronic myeloid leukemia (K562). [Method] Wild-type K562 cells were treated with different concentrations of BQ (0, 10, 20 and 40 μmol / L). MTT assay was used to detect the inhibitory effect of BQ on the proliferation of the cells. Western blot was used to detect the expression of G6PD . To construct G6PD-RNA interference lentivirus, and to infect wild type K562 cell line. The wild-type K562 cells transfected with empty vector were used as negative control cells, and the G6PD gene was detected by real-time PCR Of m RNA expression. G6PD-deficient K562-WT cells (K562-G6PD △) and negative control cells were treated with different concentrations of BQ to detect the cell proliferation inhibition. The levels of reduced glutathione (GSH) and oxidized glutathione Glutathione (GSSG) levels. [Result] The results of MTT showed that the relative proliferation rates of wild type K562 cells were significantly decreased after treated with 10, 20, 40 micromol / L BQ for 24, 48 and 72 h compared with 0 μmol / L BQ group (P <0.05 ). Western blot results showed that G6PD protein content increased with the increase of BQ concentration (r = 0.809, P = 0.008) at low dose (BQ concentration <40μmol / L); when BQ concentration increased to 40μmol / L, G6PD Protein content decreased, but still higher than BQ concentration 0μmol / L group (P <0.05). The results of real-time PCR showed that the expression of G6PD m RNA in K562-G6PD △ cells decreased by 86.65% compared with the negative control, indicating that the K562-G6PD △ cells were successfully constructed. After intervention with G6PD, the MTT results showed that the relative proliferation rate of K562-G6PD △ cells was significantly lower than that of negative control cells (P <0.05) after exposure to different concentrations of BQ. The results of colorimetric assay showed that the concentration of GSH in K562-G6PD △ cells decreased significantly when the concentration of BQ was 20μmol / L and 40μmol / L, but decreased significantly when the concentration of BQ was 40μmol / L in negative control cells. Compared with the negative control cells, the concentration of GSSG in K562-G6PD △ cells was significantly increased (P <0.05) at the concentration of 10μmol / L. [Conclusion] The proliferation of wild type K562 cells was inhibited after exposed to BQ, and the oxidation products increased. At the same time, the oxidative injury of G6PD could be resisted by activating the antioxidant system such as G6PD. After G6PD deficiency, G6PD could not be activated in Exposed to lower doses of BQ can make intracellular GSH depletion lead to accumulation of GSSG within the cell inhibition of cell proliferation increased toxicity.